Bottom Line:
We provide evidence that protocadherin genes that do not contain an NRSE in their 5' intergenic region are regulated by NRSEs in the regulatory region of their neighboring genes.We also show that protocadherin clusters in other vertebrates such as elephant shark, zebrafish, coelacanth, lizard, mouse and human, contain different sets of multiple NRSEs.Taken together, our data suggest that the neuronal specificity of protocadherin cluster genes in vertebrates is regulated by the NRSE-NRSF/REST system.

ABSTRACTThe clustered protocadherins are a subfamily of neuronal cell adhesion molecules that play an important role in development of the nervous systems in vertebrates. The clustered protocadherin genes exhibit complex expression patterns in the central nervous system. In this study, we have investigated the molecular mechanism underlying neuronal expression of protocadherin genes using the protocadherin gene cluster in fugu as a model. By in silico prediction, we identified multiple neuron-restrictive silencer elements (NRSEs) scattered in the fugu protocadherin cluster and demonstrated that these elements bind specifically to NRSF/REST in vitro and in vivo. By using a transgenic Xenopus approach, we show that these NRSEs regulate neuronal specificity of protocadherin promoters by suppressing their activity in non-neuronal tissues. We provide evidence that protocadherin genes that do not contain an NRSE in their 5' intergenic region are regulated by NRSEs in the regulatory region of their neighboring genes. We also show that protocadherin clusters in other vertebrates such as elephant shark, zebrafish, coelacanth, lizard, mouse and human, contain different sets of multiple NRSEs. Taken together, our data suggest that the neuronal specificity of protocadherin cluster genes in vertebrates is regulated by the NRSE-NRSF/REST system.

Figure 5: NRSEs in the protocadherin clusters of zebrafish, human and mouse. (A) Zebrafish protocadherin clusters. The color vertical bars represent individual variable exons, whereas the black vertical bars at the 3′ end of each subcluster represent the constant exons. Red and blue ovals in the intergenic regions of the zebrafish clusters represent the canonical and non-canonical NRSEs, respectively. The dotted lines in the Pcdh2 cluster indicate undetermined sequence gaps. The consensus sequence of zebrafish canonical NRSEs is displayed as a WebLogo plot. Dr: Danio rerio. (B) EMSA for NRSEs located in the protein-coding sequence of human and mouse protocadherin clusters. The consensus sequences are displayed as WebLogo plots. Upper-case letters represent the NRSE sequence, and the lower-case letters represent the flanking sequences. The asterisks indicate the guanine dinucleotide which was mutated to adenine dinucleotide in NRSE mutants. Hsα5m represents the mutant NRSE in the protein-coding sequence of the Hsα5. Hs: Homo sapiens, Mm: Mus muscus. Autoradiography of EMSA: arrows indicate the protein–oligonucleotide complex of the full-length fugu NRSF/REST (lanes 1, 3, 5 and 7) or the super-shifted complex in the presence of the anti-Myc antibodies (lanes 2, 4, 6 and 8). fN: nuclear extracts from pCMVmyc-fNRSF-transfected HEK293 cells; un: nuclear extracts from mock (pCMVmyc) transfected HEK293 cells.

Mentions:
To investigate whether the clustered protocadherin genes in other vertebrates are also regulated by the NRSE-NRSF/REST system, we first performed an in silico analysis of the zebrafish protocadherin clusters. Similar to fugu, zebrafish contains two independent protocadherin clusters, Pcdh1 and Pcdh2, that collectively contain at least 107 genes (6,55–57). However, unlike the highly degenerate fugu Pcdh1 cluster, the zebrafish Pcdh1 cluster contains all the three subclusters (α, γ and δ), with a total of 38 protocadherin genes. Our search revealed that seven out of the 38 genes in zebrafish Pcdh1 cluster and 37 out of the 69 genes in zebrafish Pcdh2 cluster each contain a single canonical NRSE motif in their 5′ intergenic regions, whereas Dr2α9 and Dr2α30 genes contain two such elements each (Figure 5A, Supplementary Table S1). In addition, we identified six non-canonical NRSEs in five zebrafish protocadherin genes (Dr1γ2, Dr1γ19, Dr1γ22, Dr1γ23 and Dr2α15) (Figure 5A, Supplementary Table S1). Thus, the NRSE-containing genes in the zebrafish protocadherin clusters account for about half (49/107) of the total number of genes in the cluster. We performed similar in silico search in the protocadherin clusters of human, mouse, lizard (58), coelacanth (56) and elephant shark (31), using the consensus sequence of the NRSE elements identified in fugu and mammalian genomes (40,41). We found that all of these vertebrates contain different numbers of NRSEs (25 in human, 35 in mouse, five in lizard, 11 in coelacanth and five in elephant shark) in their protocadherin clusters (Supplementary Table S2). To verify if the NRSEs in the protocadherin clusters of these vertebrates are functional, we tested the binding activity of a few selected non-canonical NRSEs by EMSA. We found that all the selected NRSEs can bind to NRSF/REST specifically in vitro (Figure 2B). These experiments indicate that the NRSEs in the protocadherin clusters of zebrafish and mammals are indeed functional regulatory elements and are likely to be involved in regulating neuronal expression of clustered protocadherin genes.Figure 5.

Figure 5: NRSEs in the protocadherin clusters of zebrafish, human and mouse. (A) Zebrafish protocadherin clusters. The color vertical bars represent individual variable exons, whereas the black vertical bars at the 3′ end of each subcluster represent the constant exons. Red and blue ovals in the intergenic regions of the zebrafish clusters represent the canonical and non-canonical NRSEs, respectively. The dotted lines in the Pcdh2 cluster indicate undetermined sequence gaps. The consensus sequence of zebrafish canonical NRSEs is displayed as a WebLogo plot. Dr: Danio rerio. (B) EMSA for NRSEs located in the protein-coding sequence of human and mouse protocadherin clusters. The consensus sequences are displayed as WebLogo plots. Upper-case letters represent the NRSE sequence, and the lower-case letters represent the flanking sequences. The asterisks indicate the guanine dinucleotide which was mutated to adenine dinucleotide in NRSE mutants. Hsα5m represents the mutant NRSE in the protein-coding sequence of the Hsα5. Hs: Homo sapiens, Mm: Mus muscus. Autoradiography of EMSA: arrows indicate the protein–oligonucleotide complex of the full-length fugu NRSF/REST (lanes 1, 3, 5 and 7) or the super-shifted complex in the presence of the anti-Myc antibodies (lanes 2, 4, 6 and 8). fN: nuclear extracts from pCMVmyc-fNRSF-transfected HEK293 cells; un: nuclear extracts from mock (pCMVmyc) transfected HEK293 cells.

Mentions:
To investigate whether the clustered protocadherin genes in other vertebrates are also regulated by the NRSE-NRSF/REST system, we first performed an in silico analysis of the zebrafish protocadherin clusters. Similar to fugu, zebrafish contains two independent protocadherin clusters, Pcdh1 and Pcdh2, that collectively contain at least 107 genes (6,55–57). However, unlike the highly degenerate fugu Pcdh1 cluster, the zebrafish Pcdh1 cluster contains all the three subclusters (α, γ and δ), with a total of 38 protocadherin genes. Our search revealed that seven out of the 38 genes in zebrafish Pcdh1 cluster and 37 out of the 69 genes in zebrafish Pcdh2 cluster each contain a single canonical NRSE motif in their 5′ intergenic regions, whereas Dr2α9 and Dr2α30 genes contain two such elements each (Figure 5A, Supplementary Table S1). In addition, we identified six non-canonical NRSEs in five zebrafish protocadherin genes (Dr1γ2, Dr1γ19, Dr1γ22, Dr1γ23 and Dr2α15) (Figure 5A, Supplementary Table S1). Thus, the NRSE-containing genes in the zebrafish protocadherin clusters account for about half (49/107) of the total number of genes in the cluster. We performed similar in silico search in the protocadherin clusters of human, mouse, lizard (58), coelacanth (56) and elephant shark (31), using the consensus sequence of the NRSE elements identified in fugu and mammalian genomes (40,41). We found that all of these vertebrates contain different numbers of NRSEs (25 in human, 35 in mouse, five in lizard, 11 in coelacanth and five in elephant shark) in their protocadherin clusters (Supplementary Table S2). To verify if the NRSEs in the protocadherin clusters of these vertebrates are functional, we tested the binding activity of a few selected non-canonical NRSEs by EMSA. We found that all the selected NRSEs can bind to NRSF/REST specifically in vitro (Figure 2B). These experiments indicate that the NRSEs in the protocadherin clusters of zebrafish and mammals are indeed functional regulatory elements and are likely to be involved in regulating neuronal expression of clustered protocadherin genes.Figure 5.

Bottom Line:
We provide evidence that protocadherin genes that do not contain an NRSE in their 5' intergenic region are regulated by NRSEs in the regulatory region of their neighboring genes.We also show that protocadherin clusters in other vertebrates such as elephant shark, zebrafish, coelacanth, lizard, mouse and human, contain different sets of multiple NRSEs.Taken together, our data suggest that the neuronal specificity of protocadherin cluster genes in vertebrates is regulated by the NRSE-NRSF/REST system.

ABSTRACTThe clustered protocadherins are a subfamily of neuronal cell adhesion molecules that play an important role in development of the nervous systems in vertebrates. The clustered protocadherin genes exhibit complex expression patterns in the central nervous system. In this study, we have investigated the molecular mechanism underlying neuronal expression of protocadherin genes using the protocadherin gene cluster in fugu as a model. By in silico prediction, we identified multiple neuron-restrictive silencer elements (NRSEs) scattered in the fugu protocadherin cluster and demonstrated that these elements bind specifically to NRSF/REST in vitro and in vivo. By using a transgenic Xenopus approach, we show that these NRSEs regulate neuronal specificity of protocadherin promoters by suppressing their activity in non-neuronal tissues. We provide evidence that protocadherin genes that do not contain an NRSE in their 5' intergenic region are regulated by NRSEs in the regulatory region of their neighboring genes. We also show that protocadherin clusters in other vertebrates such as elephant shark, zebrafish, coelacanth, lizard, mouse and human, contain different sets of multiple NRSEs. Taken together, our data suggest that the neuronal specificity of protocadherin cluster genes in vertebrates is regulated by the NRSE-NRSF/REST system.